Compositions and methods for enhancing healing and regeneration of bone and soft tissue

ABSTRACT

The invention features biodegradable materials, and in vitro and in vivo methods of using such compositions to promote bone and soft tissue growth and healing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation and cites the priority of U.S.application Ser. No. 16/249,748, filed 16 Jan. 2019, which is adivisional of and cites the priority of U.S. application Ser. No.14/818,662, filed 5 Aug. 2015, which is issued, and cites the priorityof U.S. App. No. 62/033,599, filed 5 Aug. 2014, each of which areincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Clinically, bone resorption in the maxillary and mandibular jaws occursafter loss of dentition. Partial edentulism affects 40% of the adultpopulation and is estimated to increase in the next 15 years to morethan 200 million individuals (Facts and Figures. 2012, American Collegeof Prosthodontics). In such cases, the bone resorption causes thealveolar ridge to decrease in width and height with a 50% loss in bonewidth occurring during the first year after a tooth is lost, two-thirdsof which occur in the initial 3 months (Schropp, L., et al., Int JPeriodontics Restorative Dent, 2003. 23(4). p. 313-23). The result ofthis is that before the patient's dentition is restored with dentalimplants, a separate procedure is required to replace this lost bonestructure. There are various surgical procedures available to graft thedeficit alveolar ridge for both height and width. To do this a bonegraft, commonly allograft bone powder/particulate or block is placed inthe void space to provide osteoconductive/osteoinductive cues fortargeted bone regeneration. Many of these procedures utilize a guidedbone regenerative (GBR) membrane to maintain the bone graft in place aswell as soft tissues. To date, the “ideal” GBR membrane for largedefect, alveolar ridge bone grafting has yet to be developed (Bottino,M. C., et al., Dent Mater, 2012. 28(7): p. 703-21; Dimitriou, R., etal., BMC Med, 2012. 10: p. 81).

Current biomaterials used as membrane barriers in dental extractions areoften difficult to handle, degrade quickly, and offer no enhanced woundregeneration which is paramount for complete and timely closure of thetissue over a bone graft. There is an urgent need for a biodegradablematerial that would support bone growth, promote bone and soft tissuehealing, and inhibit infection. Such a material would be useful fortreating injuries, conditions and disorders affecting bone and softtissue.

SUMMARY OF THE INVENTION

As described herein, the present invention features biodegradablebarrier materials and in vitro and in vivo methods of using suchmaterials to promote bone and soft tissue growth and healing.

In one aspect, the invention provides a composition comprising.

a) a biodegradable polymer; and

b) a honey.

In certain embodiments, the composition additionally comprises c) afiller.

In certain embodiments, the biodegradable polymer comprises a protein.In certain embodiments, the protein is gelatin. In certain embodiments,the protein is collagen.

In certain embodiments, the biodegradable polymer comprises poly(lacticacid).

In certain embodiments, the honey is present in an amount of about 1part to about 300 parts by weight relative to 100 parts by weight of thebiodegradable polymer, e.g. gelatin.

In certain embodiments, the honey is present in an amount of about 1part to about 100 parts by weight, of about 1 part to about 50 parts byweight, of about 1 part to about 15 parts by weight, or particularly ofabout 5 part to about 10 parts by weight relative to 100 parts by weightof the biodegradable polymer, e.g. gelatin.

In certain embodiments, the filler is present in an amount of 1-300parts by weight relative to 100 parts by weight of the biodegradablepolymer. Preferably, the filler is present in an amount of about 1-100parts by weight, 5-50 parts by weight or particularly 10-20 parts byweight.

In certain embodiments, the filler comprises a nanofiller, a microfilleror mixtures thereof. The nanofiller has an average diameter in nanoscaleranging from about 1 nm to about 999 nm, or less than about 1 μm. Incertain embodiments, the nanofiller suitably has an average diameterless than about 990 nm, less than about 900 nm, less than about 800 nm,less than about 700 nm, less than about 600 nm, less than about 500 nm,less than about 400 nm, less than about 300 nm, less than about 200 nm,or less than about 100 nm. In certain embodiments, the nanofillersuitably has an average diameter of about 1-100 nm, of about 10-80 nm,of about 25-75 nm, or particularly of about 50 nm. The microfiller is amicron-sized filler having an average diameter in microscale at leastabout 1 μm. The microfiller suitably has an average diameter of aboutless than about 10 μm, less than about 9 μm, less than about 8 μm, lessthan about 7 μm, less than about 6 μm, less than about 5 μm, less thanabout 4 μm, less than about 3 μm, less than about 2 μm, or particularlyof about 1-2 μm.

In certain embodiments, the filler comprises chitin whiskers. In certainembodiments, the filler comprises hydroxyapatite. In certainembodiments, the filler (such as chitin whiskers) are present in anamount of about 15 parts by weight relative to 100 parts by weight ofthe biodegradable polymer. In certain embodiments, the chitin whiskershave an average diameter of about 25-75 nm, or particularly an averagediameter of about 50 nm. In certain embodiments, the chitin whiskershave an average length of about 200-400 nm, of about 250-300 nm, orparticularly of about 280 nm.

In certain embodiments, the composition further comprises at least oneor more additional filler or at least one or more therapeutic agents,such as antibiotic. In certain embodiment, the therapeutic agent is atherapeutically effective amount of honey. In particular embodiments,the composition further comprises an antibacterially-effective amount ofhoney, which ranges from about 50 parts to about 300 parts, or fromabout 100 parts to about 200 parts by weight relative to 100 parts byweight of the biodegradable polymer. In particular embodiment, thecomposition further comprises an effective amount of honey forstimulating or enhancing regeneration (cell proliferation andmigration), which ranges from about 10 parts to about 100 parts, fromabout 20 parts to about 70 parts by weight, or particularly of about 50part by weight relative to 100 parts by weight of the biodegradablepolymer. The honey for therapeutic use is same to or different from theabove described honey.

In another aspect, the invention provides a membrane comprising:

a) a biodegradable polymer;

b) a honey.

In certain embodiments, the membrane may additionally comprise a filler.

In another aspect, the multiple-layer membrane comprising at least twolayers of a membrane of the invention.

In certain embodiments, the multiple-layer membrane comprises 2-4 layersof the membrane of the invention. In certain embodiments, themultiple-layer membrane comprises four layers of the membrane. Incertain embodiments, the at least two layers are crosslinked. In certainembodiments, the at least two layers are crosslinked with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, genipin, glutaraldehydeor mixture thereof.

In another aspect, the invention provides a method of making acomposition of the invention (i.e., a composition comprising abiodegradable polymer and a honey. The composition may additionallyinclude a filler. The method comprises: forming a composition bycombining the biodegradable polymer and honey with a solvent.

Preferably, the method comprises:

a) dispersing the filler in a solvent to form a dispersion; and

b) combining the biodegradable polymer and honey with the dispersion toform the composition.

In certain embodiments, the solvent is 2,2,2-trifluoroethanol,1,1,1,3,3,3-hexafluoro-2-propanol (HFP) or 9:1 acetic acid:water. Incertain embodiments, the solvent does not significantly solubilize thehoney under the conditions used to form and process the composition,fiber, and/or membrane.

In another aspect, the invention provides a fiber comprising:

a) a biodegradable polymer; and

b) a honey.

The fiber may further comprise a filler.

In a preferred aspect, the “fiber” may include a nanofiber, amicrofiber, or a nano-microfiber. The fiber may be formed in a bundlewithout limitation to the number or the total thickness thereof,comprising the nanofiber, the microfiber, the nano-microfiber or mixturethereof. In certain embodiments, the nanofiber has an average diameteror thickness in nanoscale ranging from about 1 nm to about 950 nm.Preferably, the nanofiber suitably has an average diameter or athickness less than about 100 nm. The microfiber has an average diameteror thickness in microscale ranging from about 1 μm to about 950 μm.Preferably, the microfiber suitably has an average diameter or athickness of about less than about 10 μm. Further, the nano-microfibersuitably has an average diameter or thickness ranging from about 100 nmto about 10 μm.

In another aspect, the invention provides a method of making a fibercomprising: a biodegradable polymer and a honey. The fiber mayadditionally comprise a filler. The method comprises:

forming a composition by combining the biodegradable polymer and honeywith a solvent; and

electrospinning the composition to form the fiber.

Preferably, the method comprises:

a) dispersing the filler in a solvent to form a dispersion:

b) combining the biodegradable polymer and honey with the dispersion toform a composition; and

c) electrospinning the composition to form the fiber.

In another aspect, the invention provides a method of making a membranecomprising: a biodegradable polymer and a honey. The membrane mayadditionally comprise a filler. The method comprises:

forming a composition by combining the biodegradable polymer and honeywith a solvent; and

electrospinning the composition to form fibers, thereby forming themembrane.

Preferably, the method comprises:

a) dispersing the filler in a solvent to form a dispersion:

b) combining the biodegradable polymer and honey with the dispersion toform a composition; and

c) electrospinning the composition to form fibers, thereby forming themembrane.

In another aspect, the invention provides a method of making a membraneof the invention, the method comprising:

a) dispersing the filler in a solvent to form a dispersion:

b) combining the biodegradable polymer and honey with the dispersion:

c) removing solvent from the dispersion to form a sponge; and

d) compressing the sponge to form the membrane.

In certain embodiments, the step of compressing comprises compressingthe sponge at a pressure of at least 3000 pounds.

In certain embodiments, the membrane is further processed to form ablock, a particulate, swelling membrane, non-compressed membrane orcompressed membrane.

In another aspect, the invention provides a multiple-layer membranecomprising:

a) a biodegradable polymer; and

b) a honey.

The multiple-layer membrane may further comprise a filler.

In another aspect, the invention provides a method of making amultiple-layer membrane of the invention, the method comprising:

forming a composition by combining the biodegradable polymer and honeywith a solvent;

electrospinning the composition to form fibers;

collecting the fibers to form at least two non-woven mesh membranes; and

attaching the at least two non-woven mesh membranes to form themultiple-layer membrane.

Preferably, the method comprises:

a) dispersing the filler in a solvent to form a dispersion:

b) combining the biodegradable polymer and honey with the dispersion;

c) electrospinning the composition to form fibers:

d) collecting the fibers to form at least two non-woven mesh membranes;and

e) attaching the at least two non-woven mesh membranes to form themultiple-layer membrane.

The multi-layer membrane may be compressed or may not be compressed.

In another aspect, the invention provides a method of promoting boneregeneration, the method comprising contacting a bone surface with acomposition, fiber, compressed membrane, particulate, swelling membrane,non-compressed membrane, or multiple-layer membrane (compressed ornon-compressed) of the invention.

In another aspect, the invention provides a method of promoting healingof a bone defect, the method comprising contacting the bone defect witha composition, fiber, compressed membrane, particulate, swellingmembrane, non-compressed membrane, or multiple-layer membrane(compressed or non-compressed) of the invention.

In another aspect, the invention provides a method of preventinginfection of a bone defect, the method comprising contacting the bonedefect In another aspect, with a composition, fiber, compressedmembrane, particulate, swelling membrane, non-compressed membrane, ormultiple-layer membrane (compressed or non-compressed) of the invention.

In another aspect, the invention provides a method of promoting softtissue healing in a damaged tissue, the method comprising contacting thedamaged tissue with a composition, fiber, compressed membrane,particulate, swelling membrane, non-compressed membrane, ormultiple-layer membrane (compressed or non-compressed) of the invention.

In another aspect, the invention provides a method of promoting amacrophage response in a tissue, the method comprising contacting thetissue with a composition, fiber, compressed membrane, particulate,swelling membrane, non-compressed membrane, or multiple-layer membrane(compressed or non-compressed) of the invention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “alteration” is meant a change (increase or decrease) as detected bystandard art known methods such as those described herein. As usedherein, an alteration includes a 10%, 25%, 40%, 50% or greater change.

By “soft tissue disease or injury” is meant any disease, disorder, ortrauma that disrupts the normal function or connectivity of a softtissue or tissues.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ,including bone.

By “effective amount” or “therapeutically effective amount” is meant theamount of a composition of the invention required to provide desiredeffect or release the symptoms of a disease relative to an untreatedsubject. The effective amount of a cellular composition used to practicethe present invention for therapeutic treatment of a disease variesdepending upon the manner of administration, the age, body weight, andgeneral health of the subject. Ultimately, the attending physician orveterinarian will decide the appropriate amount and dosage regimen. Suchamount is referred to as an “therapeutically effective” amount.“Engraft” refers to the process of cellular contact and incorporationinto an existing tissue of interest (e.g., bone or soft tissue) in vivo.

By “enhancing bone healing” is meant increasing the extent of bonegrowth or healing relative to a control condition. Preferably theincrease is by at least 2-fold, 2.5-fold, 3-fold or more.

By “microscale” is meant between 100 nm and 999 μm in size. A particlethat is microscale is larger in size than a nanotube.

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

By “reference” is meant a standard or control condition.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 1 shows SEM images of non-compressed and compressed electrospungelatin+15% CW+honey scaffolds (non-crosslinked). Scale bars andmagnification at 10 μm and 2kx, respectively.

FIG. 2 shows FibraQuant™ automated fiber diameter analysis using SEMimages from FIG. 1. The above histograms show fiber size distributionalong with the mean and standard deviations, in microns.

FIGS. 3A-3B show the results of uniaxial tensile testing of compressedelectrospun membranes: A. Strain at break, B. elastic modulus.

FIGS. 4A-4B show exemplary formable hydrated 10% honey compressedmembranes.

FIG. 5 shows DAPI images of cellularized (HDFs) compressed electrospunmembranes. Scale bars and magnification at 200 μm and 10×, respectively.

FIGS. 6A-6B show DinoLite images of general gross appearance ofnon-compressed and compressed gelatin+10% CW+30 mg/mL honey sponges.

FIG. 7A shows an exemplary membrane (sponge) particulate of varioussizes.

FIG. 7B shows an exemplary sponge particular packed in a void (socket).

FIG. 7C shows an exemplary use of the particulate that is covered by thecompressed lyophilized membrane, when the particulate is packed in avoid.

FIG. 7D shows an exemplary dry lyophilized sponge compressed by hand.

FIG. 7E shows an exemplary swollen back to original size when hydrated.

FIG. 8 shows a Carver hydraulic unit used for scaffold compression.

FIG. 9 schematically illustrates steps of an exemplary mechanicaltesting method.

FIG. 10 shows SEM images of non-compressed and compressed gelatin+CW+MHmembranes, which includes scale bars and magnification at 200 μm and100×, respectively.

FIG. 11A shows a graph including Gelatin+CW+MH degradation results (BCAassay) as shown with cumulative mean release measurement.

FIG. 11B shows a graph including Gelatin+CW+MH degradation results (BCAassay) as shown with cumulative percent release measurement.

FIG. 12 shows DAPI images of cellularized (HDFs) compressedgelatin+CW+MH membranes. Scale bars and magnification at 100 μm and 10×,respectively.

FIGS. 13A-13C show exemplary formable hydrated membranes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features biodegradable polymer-based materials ormatrices (e.g., fibers or membranes) comprising honey; and in vitro andin vivo methods of using such compositions to ameliorate an injury orcondition (e.g., bone injury or trauma associated with dental surgery).

The invention is based, at least in part, on the discovery thatbiodegradable membranes comprising honey can support and promote boneand tissue growth and regeneration. In addition, the biodegradablemembranes include an antibacterially-effective amount of honey, therebyproviding an antibacterial barrier against infection and promoting asterile environment for wound healing.

Scaffolds

In general, the materials of the invention comprise a biodegradablepolymer and a honey (e.g., an antibacterial, bactericidal, and/or woundhealing amount of honey). Preferably, the materials may additionallycomprise a filler.

A variety of biodegradable polymers are known in the art. Preferredbiodegradable polymers include proteins (such as gelatin and collagen),polymers derived from naturally-occurring monomers (such as poly(lacticacid (PLA)), and polymers derived from synthetic monomers (such aspolydioxanone (PDO)). Desirably, biodegradable materials will degradeover a time period of less than a year, more preferably less than sixmonths. In general, any biodegradable polymer that is biocompatible, andcan be shaped or formed into fibers and membranes, can be employed inthe present materials. Copolymers or mixtures/blends (multi-component)of biodegradable polymers can also be employed.

Other biocompatible polymers, some of which are biodegradable, include,e.g., Such polymers include but are not limited to the following:poly(urethanes), poly(siloxanes) or silicones, poly(ethylene),poly(vinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate),poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinylalcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinylacetate), poly(ethylene glycol), poly(methacrylic acid), polylactic acid(PLA), polyglycolic acids (PGA), poly(lactide-co-glycolides) (PLGA),nylons, polyamides, polyanhydrides, poly(ethylene-co-vinyl alcohol)(EVOH), polycaprolactone, poly(vinyl acetate) (PVA), polyvinylhydroxide,poly(ethylene oxide) (PEO) and polyorthoesters or any other similarsynthetic polymers that may be developed that are biologicallycompatible. Some preferred synthetic matrix materials include PLA, PGA,copolymers of PLA and PGA, pol caprolactone, poly(ethylene-co-vinylacetate). (EVOH). PVA, and PEO. See also U.S. Pat. No. 7,374,774 (whichis incorporated herein by reference).

The term “filler”, as used herein, refers to an organic or inorganicbiocompatible material that provides structural reinforcement orrigidity to a polymer fiber, filament, or membrane. The filler may be acrystalline, a fiber, or a particle. Alternatively, the filler suitablyhas a shape of rod, fiber, sphere, oval, polyhedral crystal, and thelike, however, the shape of the filler is not particularly limitedthereto. The filler has an average diameter in nanoscale (nanofiller)ranging from about 1 nm to about 950 nm. The nanofiller suitably has anaverage diameter of about 1-100 nm, of about 10-80 nm, of about 25-75nm, or particularly of about 50 nm. Alternatively, the filler has anaverage diameter in microscale (microfiller) that is greater than atleast about 100 nm. The microfiller suitably has an average diameter ofabout less than about 10 μm, less than about 9 μm, less than about 8 μm,less than about 7 μm, less than about 6 μm, less than about 5 μm, lessthan about 4 μm, less than about 3 μm, less than about 2 μm, orparticularly less than about 1 μm. For example, the filler is ananocrystalline or fiber material and has an average diameter orthickness of less than about 100 nm, and advantageously may have anaverage length of less than about 500 nm. Advantageously, a nanofillercan possess an electrostatic charge, which may adhere to or attractgrowth factors when implanted or applied to a wound site. Examples ofnanofiller materials suitable for use in the present materials includechitin whiskers and hydroxyapatite nanocrystals. Mixtures of fillerscomprising nanofillers and microfillers can also be used withoutlimitation.

The materials of the invention further comprise honey. Any type of honeycan be used. Examples of types of honey include Manuka honey,Leptospermum Honey or buckwheat honey. Mixtures of different honeys canalso be employed. For example, Manuka honey is an active or atherapeutic Manuka honey that has a UMF rating above 10. The honey ispresent in the compositions and materials of the invention in an amounteffect to inhibit the growth or spread of bacteria, such as pathogenicbacteria. Exemplary bacteria include S. aureus, (includingmethacillin-resistant S. aureus (MRSA)), P. gingivalis, S. epidermidis,Enterococcus faecium, E. coli, P. aeruginosa, E. cloacae, and Klebsiellaoxytoca. In addition, the buckwheat honey can be included in aneffective amount for healing.

The amount of honey to be used depends in part on the nature of thewound or injury to be treated with a composition of the invention; thetype of bacterium to be inhibited; the concentration of the honey; andthe antibacterial properties of the particular honey employed. Theantibacterial, antimicrobial, and bactericidal properties of honey aredependent on various factors including the concentration ofmethylglyoxyl (MGO), Unique Manuka Factor (UMF), the presence ofadditional phenolic compounds in the honey, wound pH, pH of the honey,and osmotic pressure exerted by the honey. One of ordinary skill in theart will be able to select a suitable type and amount of honey for usein the present compositions using no more than routine experimentation.In certain embodiments, the amount of honey is 1 part to 15 parts byweight (1-15 weight percent) based on the weight amount of thebiodegradable polymer.

In preferred embodiments, a composition of the invention include 100parts by weight of a biodegradable polymer, and about 1 part to about 15parts by weight of honey. The composition may additionally comprise10-20 parts by weight of filler. Additional compounds or agents can alsobe present as described herein.

In preferred embodiments, the composition further comprises atherapeutically effective amount of honey. For example, honey in anantibacterially-effective amount is added to the composition, whichranges from about 50 parts to about 300 parts, or from about 100 partsto about 200 parts by weight relative to 100 parts by weight of thebiodegradable polymer. In addition, additional amount of honey is addedto the composition to stimulate or enhancing regeneration (cellproliferation and migration), which ranges from about 10 parts to about100 parts, from about 20 parts to about 70 parts by weight, orparticularly of about 50 part by weight relative to 100 parts by weightof the biodegradable polymer.

Methods for Preparing Compositions

Compositions comprising a biodegradable polymer, a filler, and a honeycan be prepared by any suitable method, some of which are known in theart. In general, a filler can be suspended or dispersed in a solvent(which will not substantially dissolve the filler) to form a dispersionor suspension; the biodegradable polymer and the honey are then mixedwith the dispersion or suspension to form a composition of theinvention. In certain embodiment, a therapeutically effective amount ofhoney is additionally added to the composition for antibacterial effector enhancing regeneration. In certain embodiments, the solvent is2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) or 9:1acetic acid:water. The amount of solvent used should be minimized tofacilitate electrospinning or other processing of the composition intofibers and membranes.

Methods for Preparing Fibers and Membranes

A composition comprising a biodegradable polymer, a filler, and anantibacterially-effective amount of honey can be used to prepare fibersand membranes by any suitable method, some of which are known in theart. In one embodiment, a fiber or membrane is formed byelectrospinning. Electrospinning is a known technique (see, e.g., Li etal., Biomaterials. 2005 October; 26(30):5999-6008.) and electrospinningapparatus can be purchased commercially. For example, a charged solutioncomprising, for example, a biodegradable polymer is fed through a smallopening or nozzle (usually a needle or pipette tip). Due to its charge,the solution is drawn toward a grounded collecting plate, e.g., a metalscreen, plate, or rotating mandrel, typically 5-30 cm away, as a jet.During the jet's travel, the solvent gradually evaporates, and a chargedfiber is left to accumulate on the grounded target. The charge on thefibers eventually dissipates into the surrounding environment. If thetarget is allowed to move with respect to the nozzle position, specificfiber orientations (aligned or random) can be achieved.

The compositions of the invention can be made as electrospun fibercompositions.

In one embodiment, the invention provides a method of producing amembrane, the method comprising:

a) dispersing a filler in a solvent to form a dispersion;

b) combining a biodegradable polymer and honey with the dispersion toform a composition; and

c) electrospinning the composition to form fibers, thereby forming amembrane comprising a biodegradable polymer, a filler, and anantibacterially-effective amount of honey.

In certain embodiments, the filler is added to the composition, suchthat the step a) can be omitted and the biodegradable polymer and honeycan be combined with the solvent to form a composition.

The method may further comprise adding at least one additional filler,at least one therapeutic agent, or a therapeutically effective amount ofhoney to the composition before electrospinning. The electrospunmembrane can be formed in multiple layers. For example, the compositioncan be additionally electrospun on top of one layer or other layers tocreate multiple-layer electrospun membrane.

In another embodiment, the solvent can be removed from a dispersioncomprising a biodegradable polymer, a filler, and anantibacterially-effective amount of honey to form a sponge. Solvent canbe removed by evaporation or lyophilization (freeze-drying). Thus, inone embodiment, the invention provides a method of producing a membrane,the method comprising:

a) dispersing a filler in a solvent to form a dispersion;

b) combining a biodegradable polymer and honey with the dispersion:

c) removing solvent from the dispersion to form a sponge; and

d) compressing the sponge to form a membrane comprising a biodegradablepolymer, a filler, and an antibacterially-effective amount of honey.

In certain embodiments, the filler is added to the composition, suchthat the step a) can be omitted and the biodegradable polymer and honeycan be combined with the solvent to form a composition.

The method may further comprise adding at least one additional filler,at least one therapeutic agent, or a therapeutically effective amount ofhoney to the composition.

It will be appreciated from context that the term “membrane” is usedherein to refer to a product after compression of either electrospunmats/membranes or compression of a sponge, as described herein. Thus,the “membranes” herein include both compressed fibers and compressedsponge (unless otherwise clear from context).

The sponge can be lyophilized before compressing.

In certain embodiments, the sponge (lyophilized or non-lyophilized) canbe suitably processed in a block or a particulate or ground form beforecompressing, for example, based on applications thereof depending on thebone grafting application.

Alternatively, the compressed sponge, fibers or membrane can be suitablyprocessed in a block or a particulate or ground form after compressingdepending on the bone grafting application.

Alternatively, the sponge is not compressed, or compressed with lesspressure or substantially less pressure, e.g. by hand, only to giveswelling potential (FIGS. 7D-7E).

The multiple-layer membrane can be formed by attaching the at least twomembranes.

In certain embodiments, the multiple-layer membrane is formed bycompressing multiple layers of sponges. In particular embodiment, themultiple-layer membrane is formed from multiple lyophilized sponges bycompressing multiple layers thereof. The multiple-layer membrane can becompressed or not be compressed. For example, the multiple-layermembranes can be formed by compressing multiple layers of membranesformed by any of the methods described herein. In general, compressionof 2-10 membranes (more preferably 2-4 membranes) between two surfaces(such as stainless steel plates or blocks, e.g., in a hydraulic press)at a pressure of 4,000-24,000 pounds will generally result incompression bonding of the membranes to form a multiple-layer membrane.

Alternatively, the multiple-layer membrane can be formed using multiplesolvents. In certain embodiments, at least two or more of solventshaving difference densities are used to dissolve the fillers and tocombine other components (e.g. biodegradable polymer and honey). Forexample, solutions made from the composition and different solvents arecombined, and the combined solutions may form distinct layers based onthe densities of the solvents. After removing the solvents,multiple-layered sponges and multiple-layered membrane can be prepared.The multiple-layer membrane may be compressed or may not be compressed.

The membranes can be cross-linked using cross-linking reagents. Thus, incertain embodiments, the invention provides multiple-layer membraneshaving at least two layers, wherein the at least two layers arecrosslinked, for example, to stabilize multiple-layered membranestructure. Exemplary cross-linking reagents include1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (or other carbodiimides),genipin, or glutaraldehyde. The membranes can be immersed in a solutionof the cross-linking agent (e.g., 20-40 mM) in a solvent such asethanol. When the desired amount of cross-linking has occurred, themembranes can be removed from the solution and rinsed before use.

A membrane for use in the therapeutic methods of the invention shouldhave sufficient rigidity to support the surrounding soft tissue, bemalleable at its glass transition temperature (Tg) but regain rigidityon cooling (i.e. hold shape formed in situ), and be biocompatible inthat it will promote osseointegration and not adversely affect thesurrounding soft tissue. The membrane should resorb within 6-9 months asit takes approximately 6 months for allograft bone to consolidate intonew bone in the mandible and maxilla bone graft surgeries. The membranesof the invention are flexible, moldable upon heating, maintain theirshape upon cooling, are less acidic during degradation, and the fibrousarchitecture will regulate the macrophage (MAC) response and allow forregeneration of bone and tissue (M2 MAC phenotype) versus theinflammatory (M1 MAC phenotype).

The size and thickness of a membrane of the invention can be variedaccording to the intended use. The membranes can be spun to a desiredsize, or a sponge can be cast to a desired size, followed by compressionto a desired density and thickness. For example, barrier membranes arecommonly between 0.1-0.4 mm in thickness, so the sponge can be suitablycompressed to a thickness of about 0.1-0.4 mm.

The membrane can have any shape (round, square, rectangular, irregular).In exemplary embodiments, a membrane of the invention has a width from 1to 20 mm and a length from 1 to 20 mm. In certain embodiments, amembrane is less than 1 mm in thickness, less than 0.5 mm thickness,less than 0.3 mm in thickness, or less than 100 microns in thickness.

In certain embodiments, a membrane of the invention has a strain atbreak of at least 90%, 100%, 110% or 120%. In certain embodiments, amembrane of the invention has modulus of elasticity of at least about 5mPa, or 10, 15, 20, or 25 mPa. In certain embodiments, a membrane of theinvention has a maximum compression load of at least about 0.26N.

Therapeutic and Prophylactic Applications

The present invention provides a ready supply of materials useful forameliorating conditions associated with bone or soft tissue disease orinjury. Compositions and materials of the invention are administered(e.g., directly or indirectly) to a damaged or diseased tissue or organwhere they engraft and establish functional connections with a targettissue (e.g., bone, muscle, gum, gingiva, mucous membrane, skin). In oneembodiment, a membrane of the invention enhances bone healing. Methodsfor repairing damaged tissue or organs may be carried out either invitro, in vivo, or ex vivo. In a particular embodiment, the membrane isused in a dental application, e.g., in mandible and maxilla bone graftsurgery.

In another embodiment, the invention provides a method of promoting boneregeneration, the method comprising contacting a bone surface with acomposition, fiber, compressed membrane, particulate, swelling membrane,non-compressed membrane or multiple-layer membrane (compressed ornon-compressed) of the invention. In certain embodiments, the method isa method of promoting bone regeneration after a surgical procedure onbone, including socket preservation, ridge augmentation, sinus graftingor bone grafting.

In another embodiment, the invention provides a method of promotinghealing of a bone defect, the method comprising contacting the bonedefect with a composition, fiber, compressed membrane, particulate,swelling membrane, non-compressed membrane or multiple-layer membrane(compressed or non-compressed) of the invention.

In another embodiment, the invention provides a method of preventinginfection of a bone defect, the method comprising contacting the bonedefect with a composition, fiber, membrane, particulate, swellingmembrane, non-compressed membrane or multiple-layer membrane (compressedor non-compressed) of the invention.

In still another embodiment, the invention provides a method ofpromoting soft tissue healing in a damaged tissue, the method comprisingcontacting the damaged tissue with a composition, fiber, membrane,particulate, swelling membrane, non-compressed membrane ormultiple-layer membrane (compressed or non-compressed) of the invention.

In certain embodiments of the above aspects, the method is a method ofpromoting bone regeneration after a surgical procedure on bone,including socket preservation, ridge augmentation, sinus grafting orbone grafting.

In yet another embodiment, the invention provides a method of promotinga macrophage response in a tissue, the method comprising contacting thetissue with a composition, fiber, membrane, particulate, swellingmembrane, non-compressed membrane or multiple-layer membrane (compressedor non-compressed) of the invention.

Administration

Compositions, fiber, and membranes of the invention can be provideddirectly to a tissue or organ of interest (e.g., by direct applicationto a bone or tissue surface, or by surgical implantation). A membranecan be applied to cover, surround, fill, or otherwise contact a bone ortissue defect, wound, skin/wound healing, gingival recession or surgicalsite.

If desired, expansion and differentiation agents can be provided priorto, during or after administration of the composition, fiber, ormembrane to increase, maintain, or enhance production or differentiationof cells in vivo, including bone cells from a subject's bone or from anytype of bone graft material/transplant, i.e., allogenic, xenogenic,alloplastic or genetically produced bone. Compositions of the inventioninclude pharmaceutical compositions. When administering a therapeuticcomposition or material of the present invention (e.g., a pharmaceuticalcomposition), it will generally be formulated in a unit dosage form.Additional therapeutic agents can be applied to the fibers orincorporated within fibers during fabrication.

Formulations

Compositions, fibers, membranes, or multiple-layer membranes of theinvention of the invention can be conveniently provided as sterilepreparations. In one embodiment, a composition of the invention isprovided as a liquid, liquid suspension, gel, viscous composition, orsolid composition. Liquid, gel, and viscous compositions are somewhatmore convenient to administer, especially by injection. Viscouscompositions can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cells(e.g., embryonic stem cells, neuronal progenitors, differentiatedneurons) as desired. Such compositions may be in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, dextrose, or the like. The compositionscan contain auxiliary substances such as wetting, dispersing, oremulsifying agents (e.g., methylcellulose), pH buffering agents, gellingor viscosity enhancing additives, preservatives, flavoring agents,colors, and the like, depending upon the route of administration and thepreparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICALSCIENCE”, 17th edition, 1985, incorporated herein by reference, may beconsulted to prepare suitable preparations, without undueexperimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. The compositions can be isotonic, i.e., they can have the sameosmotic pressure as blood and lacrimal fluid. The desired isotonicity ofthe compositions of this invention may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. In addition, silversalts can be used as thickening agent. See also U.S. Pat. Nos.8,367,094; 8,173,151; and 7,998,498 (which are incorporated herein byreference). The silver salts may be added to further improveantibacterial effects of the composition. The preferred concentration ofthe thickener will depend upon the agent selected. The important pointis to use an amount that will achieve the selected viscosity. Obviously,the choice of suitable carriers and other additives will depend on theexact route of administration and the nature of the particular dosageform, e.g., liquid dosage form (e.g., whether the composition is to beformulated into a solution, a suspension, gel or another liquid form,such as a time release form or liquid-filled form).

Glycerin or similar components can be added to the admixture to improvefiber and membrane flexibility.

Exemplary agents that may be delivered together with a composition,fiber, membrane, or multiple-layer membrane of the invention of theinvention include, but are not limited to, antibiotics (including. e.g.,antibacterial silver salts), analgesics, anticoagulants,immunosuppressants, the therapeutic substance is selected from the groupconsisting of anesthetics, hypnotics, sedatives, sleep inducers,antipsychotics, antidepressants, antiallergics, antianginals,antiarthritics, antiasthmatics, antidiabetics, antidiarrheal drugs,anticonvulsants, antigout drugs, antihistamines, antipruritics, emetics,antiemetics, antispasmondics, appetite suppressants, neuroactivesubstances, neurotransmitter agonists, antagonists, receptor blockers,reuptake modulators, beta-adrenergic blockers, calcium channel blockers,disulfarim, muscle relaxants, analgesics, antipyretics, stimulants,anticholinesterase agents, parasympathomimetic agents, hormones,antithrombotics, thrombolytics, immunoglobulins, hormone agonists,hormone antagonists, vitamins, antineoplastics, antacids, digestants,laxatives, cathartics, antiseptics, diuretics, disinfectants,fungicides, ectoparasiticides, antiparasitics, heavy metals, heavy metalantagonists, chelating agents, alkaloids, salts, ions, autacoids,digitalis, cardiac glycosides, antiarrhythmics, antihypertensives,vasodilators, vasoconstrictors, antimuscarinics, ganglionic stimulatingagents, ganglionic blocking agents, neuromuscular blocking agents,adrenergic nerve inhibitors, anti-oxidants, anti-inflammatories, woundcare products, antitumoral agents, antiangiogenic agents, antigenicagents, wound healing agents, plant extracts, growth factors, growthhormones, cytokines, immunoglobulins, emollients, humectants,anti-rejection drugs, spermicides, conditioners, antibacterial agents,antifungal agents, antiviral agents, tranquilizers, cholesterol-reducingdrugs, antitussives, histamine-blocking drugs and monoamine oxidaseinhibitors. Other agents include proteins such as any one or more ofactivin A, adrenomedullin, acidic FGF, basic fibroblast growth factor,angiogenin, angiopoietin-1, angiopoietin-2, angiopoietin-3,angiopoietin-4, angiostatin, angiotropin, angiotensin-2, bonemorphogenic protein 1, 2, or 3, cadherin, collagen, colony stimulatingfactor (CSF), endothelial cell-derived growth factor, endoglin,endothelin, endostatin, endothelial cell growth inhibitor, endothelialcell-viability maintaining factor, ephrins, erythropoietin, hepatocytegrowth factor, human growth hormone, TNF-alpha. TGF-beta, plateletderived endothelial cell growth factor (PD-ECGF), platelet derivedendothelial growth factor (PDGF), insulin-like growth factor-1 or -2(IGF), interleukin (IL)-1 or 8, FGF-5, fibronectin, granulocytemacrophage colony stimulating factor (GM-CSF), heart derived inhibitorof vascular cell proliferation, IFN-gamma. IFN-gamma, integrin receptor,LIF, leiomyoma-derived growth factor, MCP-1, macrophage-derived growthfactor, monocyte-derived growth factor, MMP 2, MMP3, MMP9, neuropilin,neurothelin, nitric oxide donors, nitric oxide synthase (NOS), stem cellfactor (SCF), VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF, and VEGF164.Other agents that may be delivered together with a cell of the inventioninclude one or more of LIF, bone morphogenic protein (BMP), retinoicacid, trans-retinoic acid, dexamethasone, insulin, indomethacin,fibronectin and/or 10% fetal bovine serum, or a derivative thereof.Other agents include small oligonucleotides, such as SiDNA or SiRNAincluding at least a portion of sequences to a therapeutic target.

Those skilled in the art will recognize that the polymeric components ofthe compositions should be selected to be chemically inert and will notaffect the viability or efficacy of the cell as described in the presentinvention. This will present no problem to those skilled in chemical andpharmaceutical principles, or problems can be readily avoided byreference to standard texts or by simple experiments (not involvingundue experimentation), from this disclosure and the documents citedherein.

Dosages

A composition, fiber, or membrane of this invention can be applied orimplanted in an amount effective to provide wound-healing or otherproperties. In certain embodiments, a membrane of the invention providesa barrier effective to prevent infiltration of pathogenic bacteria intothe wound site. The skilled artisan can readily determine the amount ofthe composition, fiber, or membrane of the invention to be administeredin methods of the invention. Of course, for any composition to beadministered to an animal or human, and for any particular method ofadministration, it is preferred to determine therefore, toxicity, suchas by determining the lethal dose (LD) and LD₅₀ in a suitable animalmodel e.g., rodent such as mouse; and, the dosage of the composition(s),concentration of components therein and timing of administering thecomposition(s), which elicit a suitable response. Such determinations donot require undue experimentation from the knowledge of the skilledartisan, this disclosure and the documents cited herein. And, the timefor sequential administrations can be ascertained without undueexperimentation.

Delivery Methods

Compositions of the invention (e.g., scaffolds comprising cells) can beprovided directly to a tissue or organ of interest, such as a tissuedamaged from injury or disease (e.g., by administration into the centralor peripheral nervous system). Compositions can be administered tosubjects in need thereof by a variety of administration routes. Methodsof administration, generally speaking, may be practiced using any modeof administration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects. Such modes of administrationinclude surgical engraftment or injection (e.g., intramuscular,intra-cardiac, intraocular, intracerebroventricular).

Kits

Compositions, fibers, membranes, or multiple-layer membranes of theinvention may be supplied along with additional reagents in a kit. Thekits can include instructions for the preparation of a material (such asa membrane), a treatment regime, reagents, and equipment (test tubes,reaction vessels, needles, syringes, etc.). The instructions provided ina kit according to the invention may be directed to suitable operationalparameters in the form of a label or a separate insert.

In one embodiment, compositions, fiber, membranes, or multiple-layermembranes of the invention are useful for the treatment or prevention ofinjury or disease of bone or soft tissue. The present invention providescompositions and methods of treating such injuries or diseases and/orsymptoms thereof characterized by the loss of cells, or loss of tissuestructure, function or activity. The methods of the invention compriseadministering a therapeutically effective amount of a composition,fiber, membrane, or multiple-layer membrane described herein to asubject (e.g., a mammal, such as a human). Thus, one embodiment is amethod of treating a subject suffering from or susceptible to a disease,condition or disorder characterized by the loss of cells, or loss oftissue structure, function or activity. The method includes the step ofadministering to the mammal a therapeutic amount of a characterized bythe loss of cells, or loss of tissue structure, function or activityherein sufficient to treat the disease, condition, or disorder, orsymptom thereof, under conditions such that the disease, condition, ordisorder, or symptom thereof is treated.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa composition, fiber, membrane, or multiple-layer membrane describedherein, to produce such effect. Identifying a subject in need of suchtreatment can be in the judgment of a subject or a health careprofessional and can be subjective (e.g. opinion) or objective (e.g.measurable by a test or diagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compositions herein, such as a composition,fiber, membrane, or multiple-layer membrane described herein to asubject (e.g., animal, human) in need thereof, including a mammal,particularly a human. Such treatment will be suitably administered tosubjects, particularly humans, suffering from, having, susceptible to,or at risk for a disease, disorder, or symptom thereof. Determination ofthose subjects “at risk” can be made by any objective or subjectivedetermination by a diagnostic test or opinion of a subject or healthcare provider (e.g., genetic test, enzyme or protein marker. Marker (asdefined herein), family history, and the like).

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1. Preparation of Fibers and Membranes

The purpose of this study was to engineer a membrane with antibacterialand regenerative properties that degrades within 6-12 weeks allowing forretention of the graft while promoting a more rapid closure of theoverlying tissue. To achieve this, electrospun gelatin+chitin whiskers(CW)+honey membranes were fabricated and subsequently compressed.Compressed membranes have increased handleability, are less porous, andmaintain non-compressed fiber diameter. Less porous scaffolds aredesired for this application to provide guided regeneration for tissueclosure. Furthermore, it is documented that larger fibers and theaddition of honey (antimicrobial by nature) can independently enhancethe pro-regeneration response. Chitin whiskers (CW) are an emerging,novel filler, and have been shown to reinforce both synthetic andnatural polymeric structures. The good biocompatibility andbiodegradability also make it one of the most promising fillers.

In some experiments, gelatin was dissolved in1,1,1,3,3,3-hexafluoro-2-propanol (HFP) or 9:1 acetic acid:deionized(DI) water and electrospun with MEDIHONEY® or MANUKAGARD® (0-50 wt %).Electrospinning using HFP or acetic acid:DI water as a solvent resultedin scaffolds with micron- and nano-sized fibers, respectively. Membranes(crosslinked and non-crosslinked with 25 mM1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) were compressed (one ormultiple layers) using a hydraulic press. Compressed membranes haveincreased handleability, are less porous, and maintain non-compressedfiber diameter. Less porous scaffolds are desired for this applicationto provide guided regeneration for tissue closure. Furthermore, it isdocumented that larger fibers and the addition of honey (antimicrobialby nature) can independently enhance the pro-regeneration response. Thisstudy will further analyze the regenerative response of human dermalfibroblasts seeded on composite membranes.

Materials and Methods

CWs were prepared according to Dufresne's method with minor modification(Ji, Y-L, et al. Carbohydrate Polymers, 87, 2313-2319, 2012). Thedesired amount of CWs (15 wt % of gelatin) were redispersed in2,2,2-trifluoroethanol (TFE) by ultrasonication. Gelatin (Type B) wasadded to the CW solution at 140 mg/mL. MEDIHONEY® (100% ActiveLeptospermum Honey) was then added to the gelatin+CW solution at 0, 5,10 wt % of gelatin. Solutions were mixed and incubated at 37° C.overnight to ensure the complete dissolving/mixing of all components.Solutions were loaded into a 5 mL syringe and electrospun using thefollowing parameters: 5 mL/hr, +22 kV, and 5 inch air gap distance.Fibers were collected on a 1 inch (diameter) rotating grounded stainlesssteel mandrel.

Scaffolds were compressed to create multilayer membranes with improvedmechanical integrity while maintaining the fibrous nanostructure. 4layers of the same scaffold were compressed using metal platens on ahydraulic press for 30 seconds at 4500 pounds. Non-compressed andcompressed samples of each scaffold (0, 5, 10 wt % honey) were imagedusing a scanning electron microscope (SEM) at +20 kV to observe fiberdiameter and general porosity. Fiber diameter of all non-crosslinkedscaffold types, both compressed and non-compressed, was further analyzedby calculating average fiber diameters and standard deviations usingFibraQuant™ 1.3 software (nanoScaffold Technologies, LLC).

Crosslinking of all 4-layered membranes was achieved by placing eachmembrane in a medium petri dish containing 40 mM1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC) in ethanol for 21hours at room temperature. Upon completion, the membranes were immersedin ethanol and 6 mm discs were punched and used in cell studies.

Dog-bone punches (2.71 mm wide at narrowest point with a length of 18.63mm) were used for mechanical testing. Uniaxial tensile testing wasperformed on the dog-bone samples (n=3) with a 100 N load cell,extension rate of 1 mm/s, and a 7.7 mm starting distance between grips.Modulus of elasticity and strain at break were calculated from thestress-strain output.

Clinical adaptability/formability of membranes was scored by an oralsurgeon under both dry and hydrated (0.9% NaCl for 30 minutes)conditions. COLLAPLUG® collagen membrane (Zimmer Dental) was used as acontrol since it is currently one of the membrane barrier standards forsocket preservation surgery.

Cell Viability (DAPI)

6 mm punches of the compressed membranes were disinfected directlyfollowing crosslinking via a 30 minute ethanol soak followed by three 10minute PBS washes. Human dermal fibroblasts (HDFs) were seeded on thescaffold punches (n=3) at 5,000 cells/well in a 96 well plate. Studieswere completed over 7 days with time points at 1, 3, and 7 days. Mediachanges occurred at every time point. After each time point,cellularized scaffolds were fixed in 10% buffered formalin.4′,6-diamidino-2-phenylindole (DAPI) cell nuclei staining was thenperformed. Scaffolds were imaged using an Olympus fluorescent microscopeto visualize viable cells.

Results, Discussion and Conclusion

SEM images of non-compressed and compressed electrospun gelatin+15%CW+honey, scaffolds (non-crosslinked) are shown in FIG. 1. Fiber sizedistributions are shown in FIG. 2. FIGS. 3A and 3B show the strain atbreak (3A) and modulus of elasticity (3B) measurements.

Adaptability Formability

Table 1 shows the assessment of clinical adaptability of dry andhydrated membranes having varying amounts of honey. Best membrane (wet):0% and 10% honey. Worst membrane (wet): CollaPlug control (does not holdshape, difficult to adapt). Clinical significance: compressed membraneneeds to be hydrated before use. Formability can be tailored bycompressing fewer or more layers (FIGS. 4A-4B).

TABLE 1 Clinical adaptability of dry (D) and hydrated (wet, W)compressed membranes and CollaPlug controls scored by an oral surgeon(top). 0 1 2 3 4 0% Honey D W 5% Honey W D 10% Honey D W CollaPlug W DScale 0 = cannot be formed, either brittle or tears apart 4 = can easilybe formed, maintains structure when handled

Electrospinning and Compression

FIG. 5 shows images of compressed electrospun membranes. Compressionwhile maintaining fibrous architecture and dimensions was achieved. Somefiber welding was noticed post-compression which is most likelydependent on the crystallization state of the honey. A more dehydratedscaffold (in desiccator) will result in a more crystalline honeystructure and ultimately, less non-welded fibers upon compression.

Mechanical Testing

All scaffolds failed between 90-120% strain (no significant difference).Scaffolds containing 10% honey had significantly higher modulus valuescompared to 0% honey. This was unexpected at first since intuitively,more honey would cause the scaffolds to be less rigid. It washypothesized since the mechanical testing was performed directly fromethanol that the honey was in a dehydrated (more crystalline) state,which caused the increase in modulus. Future work will incorporateglycerin and analysis of samples hydrated with PBS which will mostlikely induce a less crystalline honey architecture and result in lessstiff scaffolds.

Cell Viability

Viable cells (HDFs) were visible on the surface of every scaffold foreach time point. Visually, it is difficult to determine any differences.However, future studies will analyze cell proliferation and cellsecreted regenerative markers and extracellular matrix.

Example 2. Preparation of Sponge

Sponges were fabricated using a 30 mg/mL gelatin solution in deionizedwater and heated to 37° C. to ensure all gelatin was in solution. 10% CW(chitin whisker) was added to the gelatin solution and sonicated. 0-30mg/mL honey was then added to the gelatin+CW solution. After the honeywent into solution, 25 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)(EDC) was added to the gelatin+CW+Honey solution, immediatelytransferred to a cylindrical mold, frozen at −80° C., and lyophilized.Dry sponges were compressed at 4,500 pounds for 30 seconds using ahydraulic press.

FIGS. 6A-6B show DinoLite images of general gross appearance ofnon-compressed and compressed gelatin+10% CW+30 mg/mL honey sponges.Noncompressed: 5.5 mm thickness; compressed: 0.3 mm thickness.

Sponges can be manufactured at any size (depending on the mold) andsubsequently compressed.

Particulate is formed similarly to the lyophilized membrane with onadditional step (FIGS. 7A-7B). The particulate can be used incombination with the lyophilized membrane, as shown in FIG. 7C. Afterthe composite solution is frozen, the frozen material can be ground up(e.g. using a blender) to form something similar to “crushed ice”. Thiscrushed ice is then lyophilized overnight to form the particulate. Sincethe particulate is intended for bone regeneration, the concentration offiller (e.g. hydroxyapatite) will be increased (e.g. to 50% or more) toenhance osteoconductivity. Development and refinement of particulate canconsist of optimizing the manufacturing process to obtain fairlyconsistent particle size. This can be achieved by controlling theblending of the frozen composite to achieve the crushed ice or bycryopulverizing (in liquid nitrogen) larger lyophilized pieces intosmaller. Particle sizes can be filtered by size using sieves orequivalent technology to obtain uniform/defined particulate sizes.Multiple methods of achieving (lyophilizing the “crushed ice” versuscryopulverizing larger (mm-sized) particulate) particle size can beperformed in order to optimize particulate size. Preferably, theparticulate has a size or an average diameter ranging from about 100 μmto about 10 mm, or particularly from about 1 mm to about 5 mm.

Both dry and hydrated, compressed membranes of this composition shouldbe hydrated before use (FIG. 7E) and can be easily cut/sized withscissors and have great handleability. Upon hydration, membranes becomemore flexible and can be maneuvered within the surgery site easily uponimplantation. Once initially hydrated, the handleability alone is asignificant improvement from existing membranes such as COLLAPLUG®. Evenafter a few days of being hydrated, current natural biodegradablemembranes such as BIO-GIDE® begin to lose their mechanical integrity.

Example 3. Compressed Membrane for Bone Grafting Applications

Further, the excellent biocompatibility and biodegradability also makeit one of the most promising fillers. These compressed membranes combinethe advantages of a film-like material with a bioactive surface tofurther enhance cell response and guided tissue regeneration (GTR).Gelatin+CW+MH membranes exhibit enhanced biocompatibility andbiodegradability which suggests their use as an alternative to currentclinical products.

Methods and Materials Scaffold Fabrication

Scaffolds were fabricated using a 30 mg/mL gelatin solution. 10% CW (wt% of gelatin) were dispersed in DI water and sonicated using a microtipfor 30 seconds at 2% amplitude. Gelatin and 0, 5, or 25% MH (wt % ofgelatin) were then solubilized within the CW solution via incubation at37° C. for 1 hour. After a uniform solution was achieved, 40 mM1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) EDC cross-linker wasadded, briefly mixed, immediately transferred to a small Petri dish,frozen overnight at −80° C., and lyophilized. Lyophilized sponges werethen sliced into 4 mm thick sections and compressed using a hydraulicpress (FIG. 8) at 4500 pounds for 30 seconds to create the finalmembranes (thickness between 300-400 μm).

Degradation

Scaffold degradation via release kinetics was studied by quantifyingprotein release from each 6 mm scaffold over 14 days. Scaffolds wereincubated at 37° C. in 1×PBS with PBS replaced at each time point. After1, 4, 7, 11, and 14 days, the releasate was analyzed for general proteinusing the Pierce BCA Protein Assay. Gelatin and MH could not bedistinguished and both contributed to the quantitative cumulative meanconcentration results. To account for this, cumulative percent releasewas calculated by using fully degraded non-crosslinked scaffolds astotal initial protein content: % release=(release)/(total initialcontent)*100.

Cell Adhesion

6 mm discs of each scaffold type were loaded into 96-well plates.Current clinical membranes, GEISTLICH BIO-GIDE® (collagen) and KLSMARTIN RESORB-X®, (polylactic acid, PLA film), were punched and used asclinical controls. All membranes were disinfected (30 minutes Ethanoland three 10 minute PBS washes) prior to cell seeding. 20,000 humandermal fibroblasts (HDFs) were seeded on membranes and cultured for 14days. After 1, 7, and 14 days, media was removed and frozen whilecellularized membranes were fixed in 10% formalin. Fixed scaffolds werestained with 4′-6-diamidino-2-phenylindole (DAPI) and their cell seededsurfaces fluorescently imaged to visualize cell attachment.

Mechanical Testing

Hydrated acellular scaffolds were analyzed using a uniaxial platencompression system to determine peak load. Rectangles (2.5×0.5 cm) werecut and fixed in an arch position by anchoring the ends 1 cm apart (FIG.9). The upper platen was lowered to the scaffold surface and thefollowing parameters were used: 10 mm/min test speed and 250samples/second data acquisition rate. Compression was continuous untilthe top platen reached the anchors. Run was terminated just before thiscontact occurred and maximum force exerted by the scaffolds was recordedin Newtons (N).

Clinical Adaptability/Formability

After hydration, all gelatin+CW+MH membranes were scored by an oralsurgeon under both dry and hydrated (0.9% NaCl for 30 minutes)conditions. KLS MARTIN, BIO-GIDE and COLLAPLUG® (collagen membrane,Zimmer Dental) were used as clinical control membranes.

Discussion and Conclusion Sponges and Compression

All gelatin+CW+MH scaffolds exhibited the same non-compressed (porous)and compressed (less porous) surface architecture with no visualdiscernible differences between scaffold types (FIG. 10). The compressedsurface provides a template for GTR compared to a porous membrane wherecells initially migrate throughout the scaffold.

Degradation

The addition of 5% MH resulted in a similar concentration releaseprofile compared to 0% MH, with both beginning to plateau after 14 days(FIGS. 11A-11B). The +25% MH membranes exhibited a more linear releaseprofile over 14 days, suggesting degradation at a constant rate. After 1day, 0%, +5%, and +25% MH released 17%, 17%, and 22% of total initialcontent, respectively. After 14 days, 0%, +5%, and plus 25% MH released44%, 34%, and 49% of total initial content, respectively. The cumulativepercent release graphs revealed interesting profiles, suggesting theaddition of 5% MH slows the degradation rate of the scaffold. This wasnot expected since the addition of any amount of MH was thought toincrease the degradation rate (evident in +25% MH graph). The dataprovides insight to the tailorable degradation rates solely based on theincorporation of various concentrations of MH.

Adaptability-Formability

Clinical adaptability of dry (D) and hydrated (wet, W) compressedmembranes and Bio-Gide, KLS Martin. and CollaPlug controls scored by anoral surgeon. When hydrated, all gelatin+CW+MH membranes handledsimilarly to Bio-Gide controls with higher percentages of incorporatedMH resulting in increased membrane tearing (Table 2). However, drygelatin+CW+MH membranes had greater adaptability compared to controls.In the hands of the surgeon, compressed membranes handled similar toclinical collagen membranes (FIGS. 13A-13C).

TABLE 2 Adaptability/Formability 1 2 3 4 5 0% MH W D +5% MH W D +25% MHW D Bio-Gide W D KLS Martin D W CollaPlug W D Scale 1 = cannot beformed, brittle or tears apart 5 = can easily be formed, maintainsstructure when handled

Compression Testing

All gelatin+CW+MH membranes exerted a max force within the range of0.02-0.03 N while the Bio-Gide and KLS Martin controls exerted 0 N and0.75 N, respectively. Gelatin+CW+MH membranes show improved mechanicalproperties compared to the Bio-Gide control which would not maintain anarch for testing (Table 3). The higher KLS Martin values are expectedsince it is a non-porous PLA film.

TABLE 3 Compression Testing 0% MH +5% MH +25% MH Bio-Gide KLS Martin0.03N 0.03N 0.02N 0N 0.75N

Cell Adhesion

The addition of MH significantly increased cell attachment on day 1compared to 0% MH and Bio-Gide membranes (FIG. 12). KLS Martin membranesalso attached a high number of cells because of its 2D film surfacesimilar to tissue culture plastic. The drawback of KLS Martin (PLA) isits degradation which leads to an acidic microenvironment. After 7 and14 days, all gelatin+CW+MH membranes were covered in cells whereBio-Gide controls still had no visible cells attached. Fluorescentimaging became more difficult at 7 and 14 days most likely due to somemigration of the cells as they remodeled the membrane. Future studieswill analyze cell proliferation, viability, secreted regenerativemarkers, and extracellular matrix production.

OTHER EMBODIMENTS

From the foregoing description, it w ill be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A composition, comprising: honey; and one or morebiodegradable polymers, wherein the composition is in asurgically-implantable form, and wherein the composition promotes tissueregeneration when surgically implanted in contact with a tissue in asubject.
 2. The composition of claim 1, wherein the composition isprovided as a solid, a membrane, a multi-layer membrane, or aparticulate.
 3. The composition of claim 1, wherein the composition isdegradable in vivo.
 4. The composition of claim 1, wherein the honeycomprises methylglyoxal.
 5. The composition of claim 1, wherein the oneof more biodegradable polymers comprise at least one natural polymer. 6.The composition of claim 5, wherein the at least one natural polymercomprises collagen or gelatin.
 7. The composition of claim 1, whereinthe one of more biodegradable polymers comprise at least one syntheticpolymer.
 8. The composition of claim 7, wherein the at least onesynthetic polymer is selected from the group consisting of; apoly(urethane), a poly(siloxane) or a silicone, a poly(ethylene),poly(vinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate),poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinylalcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinylacetate), poly(ethylene glycol), poly(methacrylic acid), polylactic acid(PLA), polyglycolic acids (PGA), poly(lactide-co-glycolides) (PLGA),polydioxanone (PDO), a nylon, a polyamide, a polyanhydride,poly(ethylene-co-vinyl alcohol) (EVOH), polycaprolactone, poly(vinylacetate) (PVA), polyvinylhydroxide, poly(ethylene oxide) (PEO),polyorthoesters PEA, a copolymer of PLA, PGA, PLGA, PDO, or PEO, andcombinations thereof.
 9. The composition of claim 7, wherein the one ormore biodegradable polymers comprise at least one natural polymercomprising collagen or gelatin.
 10. The composition of claim 1, whereinthe honey is present in an amount of from 1 part to 300 parts by weightrelative to 100 parts by weight of the one or more biodegradablepolymers.
 11. A multi-layer membrane, comprising: a first layerincluding one or more biodegradable polymers; a second layer includingone or more biodegradable polymers; and honey in at least one of thefirst layer or the second layer.
 12. The multi-layer membrane of claim11, wherein at least one of the first layer or the second layer iscross-linked.
 13. The multi-layer membrane of claim 11, wherein the oneor more biodegradable polymers of the first layer comprise a naturalbiodegradable polymer.
 14. The multi-layer membrane of claim 13, whereinthe natural biodegradable polymer comprises collagen or gelatin.
 15. Themulti-layer membrane of claim 11, wherein the one or more biodegradablepolymers of the second layer comprises at least one syntheticbiodegradable polymer.
 16. The multi-layer membrane of claim 15, whereinthe synthetic biodegradable polymer is selected from the groupconsisting of: a poly(urethane), a poly(siloxane) or a silicone, apoly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxy ethylmethacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate),poly(vinyl alcohol), poly(acrylic acid), poly acrylamide,poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylicacid), polylactic acid (PLA), polyglycolic acids (PGA),poly(lactide-co-glycolides) (PLGA), polydioxanone (PDO), a nylon, apolyamide, a polyanhydride, poly(ethylene-co-vinyl alcohol) (EVOH),polycaprolactone, poly(vinyl acetate) (PVA), polyvinylhydroxide,poly(ethylene oxide) (PEO), polyorthoesters PEA, a copolymer of PLA,PGA, PLGA, PDO, or PEO, and combinations thereof.
 17. The multi-layermembrane of claim 11, further comprising a third layer including one ormore biodegradable polymers and the honey, wherein the second layer isdisposed between the first layer and the third layer.
 18. Themulti-layer membrane of claim 11, wherein the layer comprising honeypromotes tissue regeneration when surgically implanted in contact with atissue in a subject.
 19. The multi-layer membrane of claim 11, whereinthe honey comprises methylglyoxal.
 20. The multi-layer membrane of claim11, wherein the honey is present in the first layer or the second layeran amount of from 1 part to 300 parts by weight relative to 100 parts byweight of the one or more biodegradable polymers in the first layer orthe second layer, respectively.
 21. A method of promoting tissueregeneration in vivo in a subject, the method comprising: providing acomposition, the composition including honey and one or morebiodegradable polymers; and implanting the composition in contact withtissue in the subject through a surgical site of the subject, whereinthe composition promotes tissue regeneration in the tissue whencontacted with the tissue.
 22. The method of claim 21, wherein thecomposition is provided in solid form.
 23. The method of claim 21,wherein the composition is provided as a membrane, a multi-layermembrane, or a particulate.
 24. The method of claim 21, wherein the oneof more biodegradable polymers comprise at least one natural polymer.25. The method of claim 24, wherein the at least one natural polymercomprises collagen or gelatin.
 26. The method of claim 21, wherein theone of more biodegradable polymers comprise at least one syntheticpolymer.
 27. The method of claim 26, wherein the at least one syntheticpolymer is selected from the group consisting of: a poly(urethane), apoly(siloxane) or a silicone, a poly(ethylene), poly(vinyl pyrrolidone),poly(2-hydroxy ethyl methacrylate), poly(N-vinyl pyrrolidone),poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid),polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol),poly(methacrylic acid), polylactic acid (PLA), polyglycolic acids (PGA),poly(lactide-co-glycolides) (PLGA), polydioxanone (PDO), a nylon, apolyamide, a polyanhydride, poly(ethylene-co-vinyl alcohol) (EVOH),polycaprolactone, poly(vinyl acetate) (PVA), polyvinylhydroxide,poly(ethylene oxide) (PEO), polyorthoesters PEA, a copolymer of PLA,PGA, PLGA, PDO, or PEO, and combinations thereof.
 28. The method ofclaim 26, wherein the one or more biodegradable polymers comprise atleast one natural polymer comprising collagen or gelatin.
 29. The methodof claim 21, wherein the honey is present in an amount of from 1 part to300 parts by weight relative to 100 parts by weight of the one or morebiodegradable polymers.
 30. The method of claim 29, wherein the honeycomprises methylglyoxal.